Localization of Chitin Synthase Activity in Subcellular Fractions of Schizophyllum commune Protoplasts

Abstract

SUMMARY: Plasma membranes of Schizophyllum commune were stabilized against fragmentation by coating protoplasts with concanavalin A (Con A). A uniform distribution of Con A over the membrane was demonstrated cytochemically. After lysis of the protoplasts in the presence of an inhibitor of proteolysis, plasma membranes were purified and, together with other subcellular fractions, assayed for chitin synthase activity. About 50 % of the chitin synthase activity was associated with the plasma membranes and largely occurred in an active form. About 30% of the chitin synthase, in an inactive form, was recovered in fractions sedimenting at 40000 to 300000 g. The product of the plasma membrane-bound enzyme was shown to be α-chitin occurring as microfibrils (about 6 nm wide). The chitin synthase could not be detached from the plasma membranes by incubation with substrate and activator at 0 0C and gradient centrifugation showed that the synthesized chitin remained associated with the plasma membranes.